- Email: [email protected]
Effects of galanin on the opossum internal anal sphincter: Structure-activity relationship
GASTROENTEROLOGY 1991;100:711-718
Effects of Galanin on the Opossum Internal Anal Sphincter: Structure-Activity Relationship S U S H A N T A CHAKDER a n d S A T I S H R A T T A N Division of Gastroenterologyand Hepatology,Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania
The present study was carried out to investigate the effects of porcine galanin-(1-29), N-terminal fragment galanin-(1-10), C-terminal fragment galanin(15-29), and the middle fragment galanin-(7-16) on the spontaneous tension of the opossum internal anal sphincter and on the decrease in the resting internal anal sphincter tension in response to neural stimulation by electrical field stimulation. Galanin and galanin-(1-10) caused a concentration-dependent decrease in the resting tension of internal anal sphincter and an augmentation of the percent decrease in the resting tension with electrical field stimulation. Galanin-(15-29), on the other hand, produced an increase in the resting tension of the internal anal sphincter and had no effect on the electrical field stimulation-induced decrease in the resting tension. Galanin-(7-16) produced no significant effect on the internal anal sphincter. The decrease in the internal anal sphincter tension b y galanin and galanin-(1-10) was partially antagonized by tetrodotoxin, whereas the increase in the internal anal sphincter tension caused by galanin(15-29) was not modified by tetrodotoxin. In contrast to its effect in the internal anal sphincter, galanin caused an increase in the resting tension and suppressed a decrease in the lower esophageal sphincter tension in response to electrical field stimulation. From these findings we conclude that (a) galanin exerts an inhibitory effect on.the internal anal sphincter by activating galanin receptors both at the intramural inhibitory neurons and at the internal anal sphincter smooth muscle and that the N-terminal portion of galanin m a y be responsible for these actions; (b) the contractile dction of galanin is produced by its action on the smooth muscle; and (c) the actions ofgalanin on the gastrointestinal tract are tissue s p e c i f i c .
alanin, a new neuropeptide with 29 amino acids, has been shown to be present in the central as G well as in the peripheral nervous system (1-3). In the gut, galanin has been shown to be widely distributed from the esophagus to the large intestine (4-9). It has been suggested that galanin is present in the myenteric and the Meissner's plexuses and" in the smooth muscle layers of the gut (6,7,9,10). Furthermore, galanin-immunoreactive fibers have been shown to be present in close proximity of the smooth muscle cells of the gut (10). Despite its wide distribution, the exact role of galanin in the gut is not clearly understood. It has been observed that galanin exerts both excitatory (1,5,11-14) and inhibitory effects on the motor functions of the gut (13-17). These actions may be the result of either a direct effect or may occur indirectly via the release of another neurotransmitter substance. Galanin has been suggested to be an excitatory neurotransmitter for certain components of the longitudinal smooth muscle contraction of the rat ileum (18). In the canine small intestine (15) and the pyloric sphincter (16), galanin has been suggested to act as an inhibitory neurotransmitter. Thus, galanin has been shown to have different effects on different parts of the gut. In some cases, the in vivo effects are different from those observed in vitro studies (19). However, the in vitro effects of galanin on the spontaneously tonic smooth muscles have not been examined. Recently, galanin immunoreactivity has also been demonstrated in the region of the canine internal anal sphincter where it coexists with vasoactive intestinal
Abbreviations used in this paper: IAS, internal anal sphincter; EFS, electrical field stimulation; TFX, tetrodotoxin. © 1991 by the American Gastroenterological Association 0016-5085/91/$3.00
712 CHAKDERAND RATTAN
p o l y p e p t i d e (VIP) [8). Previous studies on the internal anal s p h i n c t e r (/AS) suggest that VIP is one of the inhibitory n e u r o ~ a n s m i t t e r s ; also, the possibility of i n v o l v e m e n t of m u l t i p l e inhibitory n e u r o t r a n s m i t t e r s in the IAS has b e e n raised (20,21). T h e role of galanin in t h e / A S , however, is not k n o w n . Consequently, the p u r p o s e of the p r e s e n t investigation w a s to s t u d y the influence of galanin and its fragments o n the resting tone of the /AS a n d on neurally m e d i a t e d IAS relaxation. M a t e r i a l s a n d Methods
Preparation of Smooth Muscle Strips Opossums of either sex were killed by exsanguination after pentobarbital anesthesia (40 mg/kg IP). The entire anal canal along with a part of the rectum was isolated and transferred to a dissecting tray containing oxygenated I(rebs physiological solution. The composition of the Krebs solution (in mmolJL) was as follows: NaCI, 118.07; KCI, 4.69; CaC12, 2.52; MgSO 4, 1.16; NaH2PO 4, 1.01; NaHCO3, 25; and glucose, 11.10. • The anal canal was freed from extraneous tissues of blood vessels, nerves, connective tissue, fascia, and the external anal sphincter skeletal muscle by sharp dissection. The anal canal was then opened by an incision along the longitudinal axis and pinned flat on the dissecting tray with the mucosal side up. The mucosa was removed by gentle scraping with fine scissors and forceps, starting from the uppermost part of the anal canal. Circular smooth muscle strips (approximately 2 mm wide and 1 cm long) were obtained from the circumference of the anal canal. The ends of the muscle strips were tied with 4-0 silk suture.
Recording of Isometric Tension The smooth muscle strips were transferred to jacketed 2-mL tissue baths filled with Krebs solution, which was maintained at 34°C and equilibrated with 95% O2 5% CO2, and were anchored to the muscle bath at the lower end and isometric muscle transducers (model FTO3; Grass Instruments Co., Quincy, MA) at the other end. The isometric tension of the smooth muscle strips at rest as Well as that in response to different stimuli were recorded on a Beckman Dynograph Recorder (model R411; Beckman Instruments, Schiller Park, IL). After an equilibration period of 1 hour, the optimal length and the baseline of each /AS smooth muscle strip was determined according to a previously described method (22). The smooth muscle strips of the lower esophageal sphincter (LES) were prepared in the same manner according to a previously described method (23).
Electrical Field Stimulation Once the plateau of spontaneous/AS or LES tension was achieved, the resting tension of each smooth muscle strip was determined by supramaximal electrical field stim-
GASTROENTEROLOGYVol. 100, No. 3
ulation (EFS) (30 V, 20 Hz, 0.5 ms pulse duration, for 4 seconds). The response to EFS (20-30 V, 0.5 n~ pulse duration, for 4 seconds) at varying frequencies (0.5-5 Hz) on the resting tension of the/AS strips in the presence and absence of various agents was examined. The electrodes used consisted of thin platinum wires lying parallel to the smooth muscle strips; the EFS was delivered by a Grass stimulator (model $48; Grass Instruments).
Drug Responses The effect of different agents on the resting tension of the IAS and on the decrease in the/AS tension by EFS was investigated. The response to different concentrations of the agonists on the resting IAS tension was examined using cumulative concentration responses (24). After the concentration-response curves had been derived, the smooth muscle strips were washed six times, the resting tension was allowed to recover to the pretreatment level, and an additional 30-minute rest was given before the next agonist concentration-response curve was derived. The responses to different agents and stimuli that caused a decrease in the resting tension of the IAS were quantified as percentage of maximal decrease in the/AS tension by supramaximal EFS. The responses of agents which caused an increase in the [AS tension were quantified as the percentage of maximal rise in the tension by the maximal concentration ofbethanechol (1 x 10 -~ mol/L). In the case of antagonists or the neurotoxin, tetrodotoxin, a precalculated amount of the agent was added in the bath to achieve the desired final concentration. The concentrations of the different antagonists used have been previously tested to be appropriate against their respective agonists and stimuli. The concentration-response curves of the respective agonists and the frequancy-response curves were examined before and after the addition of galanin and its fragments.
Drugs The following drugs were used: galanin (porcine, mol wt 3211.03) (Peninsula Laboratories, Inc., Belmont, CA]; galanin 1-10 (mol wt 1081.31): galanin-(7-161 (mol wt 1011.31); galanin-(15-29) (mol wt 1711.09) (synthesized in the Protein Chemistry Laboratory, University of Florida, Gainesville, FL); tetrodotoxin (TTX, mol wt 319.28) (Calbiochem, San Diego, CA); propranolol hydrochloride (tool wt 292.34) (Ayerst Laboratories, New York, NY); atropine sulphate (mol wt 694.82); hexamethoninm chloride (mol wt 273.28); and VIP (porcine, mol wt 3326) (Sigma Chemical Co., St. Louis, MO). All agents were dissolved and diluted in Krebs solution. The solutions of different agents used were freshly prepared on the day of the experiment, and the volume of solutions added to the muscle baths was not allowed to exceed 0.1 mL. The vials, glassware, pipette tips, and the muscle baths were pretreated with 2.5% bovine serum albumin.
March 1991
MOTOR ACTIONS OF GALANIN 713
Data Analysis All changes in the resting (AS tension in response to
100
drugs and EFS were expressed on the bases of percent and absolute changes in a given smooth muscle strip before find after different pretreatments. All the values were expressed as mean -+ SE of different experiments. For comparison purposes, the responses to the agonists and EFS, before and after the use of antagonists, were obtained from the same smooth muscle strip. Thus each [AS smooth muscle strip served as its own control. The statistical significance of group differences was analyzed using Student's t test (25).
Z W F--
+/
W
Z W (.~ n~ w o_
Effects of Galanin and its Fragments on the Resting Tension of Internal Anal Sphincter
Tetrodotoxin (1 x 10 -6 tool/L), which abolished the decrease in the IAS tension in response to
]
60
Results
Influence of Tetrodotoxin on the Inhibitory Effects of Galanin on the Resting Tension of Internal Anal Sphb~cter
80
Z
n~
The amino acid sequences of galanin and the different fragments used in the present study are given in Table 1. Galanin and the N-terminal fragment, galanin-(110), caused a concentration-dependent decrease in the resting tension of the IAS. On the other hand, galanin-(15-29), the C-termfnal fragment of galanin, caused an increase in the IAS tension (Figures 1 and 2). The maximal decrease in the I_AStension observed with galanin (3 x 10 -~ mol/L) was 51% + 3%. The maximal decrease with the highest concentration of galanin-(1-10) tested (1 x 10 -4 mol/L) was only 24% _+ 2%. Higher concentrations of galanin-(1-10) were not tested because of solubility problems. The middle fragment of the galanin molecule, galanin-(7-16), produced variable effects on the resting tension of the I.AS. Of the seven smooth muscle strips examined, four produced no response, two showed contraction, and in one the response was biphasic (relaxation followed by contraction).
A - - A GALANIN-(I5-29)
Z 0
40
+/
O ~ O ~ Z 0 oo Z w
O
-20 ~T
~O 0
o~
-40
~o~ 6
Z ._J _J F-Z W C.) nl W 8_
A
A
,/
20
If', A
O--O CALANIN
-60
e--o
0ALANIN-0-10)
-80 -100
//
I
-7
l
I
,
-6
I
-5
~
I
-4
LOG [AGONIST] M Figure 1. Concentration-effect curves showing the effects ofgalanin and its different fragments. Each point represents mean -- SE of four observations obtained from different animals. The absence of the SE bars in certain points in this and the subsequent figures is caused by their small size and loss in the symbols in computerized graphs. Galanin and galanin-(1-10l caused a concentrationdependent decrease in the resting tension of the IAS. Galanin(15-29), on the other hand, caused a concentration-dependent increase in the resting tension of the [AS. Galanin-(7-16) had no reproducible effect on the resting IAS tension (not shown in the figure).
Table 1. Amino Acid Sequences of Galanin and Its Fragments Galanin
G•y-Trp•Thr-Leu-Asn•Ser-A•a•G•y-Tyr•Leu-Leu•G•y•Pr••His•A•a-lIe-Asp-Asn•His•Arg-Ser-Phe•His-Asp-Lys-Tyr•G•y-Leu-A•a•NH Galanin-(1-10)
Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu Galanin-(7-16)
Ala-Gly-Tyr-L[u-Leu-Gly-Pro-His-Ala-Ile Galanin-(15-29)
Ala-Ile-Asp-Asn-His-Axg-Ser-Phe-His-Asp-Lys-Tyr-Gly-Leu-Ala-NH2
2
714
CHAKDER AND RATTAN
GASTROENTEROLOGY Vol. 100, No. 3
Galanin
2_
(M)
2
o ~
o
~
•
•
•
•
o
,
&
Galanin-(I-10)
3 ,nin f
control
~0
• GO).
'
"~,
(M) I
,~
I
.5
I
EFS
I
I
2
5
(I-:IZ)
Galanin ~
Ot
•
Galanin-(I 5-29)
Figure 2. Typical responses of galanin a n d its different fragments on the resting tension of the [AS (left) and on the decrease in the [AS tens i o n in r e s p o n s e to EFS (right). Galanin and galanin(1-10) caused a concentration-dependent decrease, whereas galanin-(15-29) caused an increase in the [AS tension.
(M)
.~..4
0 .5 1
OL-.
EFS
:i:ii" _
,
2
5
(HZ)
--
neural stimulation, caused partial but significant ddcrease in the inhibitory responses to galanin (Figure 3). The inhibitory response of galanin on the IAS were not modified by VIP or calcitonin gene-related peptide (CGRP) antagonists (unpublished observations). As with galanin, the decrease in the resting tension of the IAS produced by galanin-(1-10) was also partially but significantly suppressed by the neurotoxin. The decrease in the resting tension of the IAS with I x 10 -s and 3 x 10 -s mol/L of galanin-(1-10)in the control experiments was 12% _+ 2% and 19% _ 2%, respectively. In the presence of TTX these values were 5% _ 1% and 9% _ 2%, respectively (P < 0.05 for both; n = 4).
Influence of Atropine, Hexamethonium, and Propranolol on the Inhibitory Effects of Galanin on the Resting Tension of the Internal Anal Sphincter The muscarinic antagonist atropine (1 x 10 -s mol/L) had no significant influence on the inhibitory effects of galanin and galanin-(1-10) on the resting tension of the IAS smooth mt~scle strips (Figure 4).
ELECTRICAL FIELD STIMULATION [HZ] 0.5 0 ~/J--
"~
z
1 .L
~,~
2 ~
5 .1.
, --
10 3.
== EFS (l'l'X)
~(5 "e
o
\o~\i
\6
-20
,,,z 1-03 ~-- - 4 0 z
-_.I ,_I
fi
OALANIN (TI'X)
X ~
0"--(5
OALANIN (CONTROL)
T n
-6o
z w
[]
(3wn.-80 0..
n
-100
/i/
I -7
,
EFS (CONTROL)
I , ~x13 , -6 -5 LOG [GALANIN] M
I -4
Figure 3. Influence of Trx on the inhibitory responses ofgalanin on the IAS. Tetrodotoxin in a concentration that abolished EFS-mediated decrease in the [AS tension resulted in a significant antagonism of the decrease in the IAS tension caused by galanin ( P < o.os; n = 4).
March 1991
MOTOR ACTIONS OF GALANIN
GALANIN
GALANIN-(I-IO)
Ox~O-eM) (3XlO-eM) OXlO-~M) (3Xlo-SM)
1
Z
o Z W I--
_<
-20
Z _1 ._1
~_ - 4 0 Z W
CONTROL ATROPINE (1X10-5M)
(3 W
-60 Figure 4. Influence of atropine on the decrease in the IAS tension caused by galanin and galanin-(1-10). Atropine failed to cause a
significant modification of the responses of galanin and gnlanin(1-10) (P > 0.05, n = 4).
The nicotinic antagonist hexamethonium (1 x 10 -6 mol/L), and ~-adrenoceptor antagonist propranolol (1 x 10 -6 mol/L) also had no significant effect on the decrease in the [AS tension caused by galanin. In this series of experiments, the decrease in the resting tension of the [AS with 1 x 10 -6 and 3 x 10 -6 mol/L galanin was 14% _+ 2% and 31% -+ 4%, respectively, in the controls; after the administration of hexamethonium and propranolol, the corresponding values were 13% -+ 2% and 30% -+ 3% and 11% - 2% and 29% +6%, respectively. These values were not significantly different from the control values (P > 0.05; n = 4).
mol/L galanin. Interestingly, the percent decrease in the resting tension in response to EFS in the presence of galanin was significantly greater than the decrease during control EFS. The quantitative data is shown in Figure 5. Galanin (3 x 10 -7 tool/L) caused a significant augmentation in the decrease in the [AS tension caused by 0.5 and 1 Hz of EFS from 42% _+ 2% and 59% _+ 5% to 68% +- 5% and 79% _ 4%, respectively, (P < 0.05; n = 4). The resting tension in these experiments before and after galanin was 1.85 _+ 0.28 and 1.50 _ 0.15 g, respectively. Galanin-(1-10) had an effect similar to galanin in causing an augmentation of EFS-induced decrease in the resting tension of the IAS smooth muscle (Figure 6). However, galanin-(15-29) and galanin-(7-16) had no significant effect on the decrease in the resting [AS tension in response to EFS (Figure 6). An actual tracing showing augmentation of the [AS responses to EFS by galanin has been included in Figure 2.
Influence of Galanin on the Decrease in the Resting Tension of the Internal Anal Sphincter Smooth Muscle by Vasoactive Intestinal Polypeptide To test the possibility that the augmentation of neurally mediated [AS relaxation is via the potentiation of the inhibitory effect of the released inhibitory neurotransmitter, we tested the influence of galanin on the percent decrease in the [AS tension by one of the candidates of inhibitory neurotransmitters, VIP. Galanin did not cause any significant change in VIP-induced decrease in the resting tension of the ELECTRICAL FIELD STIMULATION [HZ]
Influence of Tetrodotoxin and Atropine on the Increase in the Internal Anal Sphincter Tension Caused by Galanin-(15-29) The neurotoxin TTX and the muscarinic antagonist atropine had no significant effect on the resting tension of the [AS smooth muscle. The increase in the [AS tension with galanin-(15-29), 3 x 10 -6 mol/L and 3 x 10 -s mol/L, was 27% +- 6% and 53% _ 7% before and 23% - 8% and 41% _ 8% after TTX, respectively, (P > 0 . 0 5 ; n = 5).
Influence of Galanin and Galanin Fragments on the Relaxation of the Internal Anal Sphincter Smooth Muscles by Neural Stimulation In this set of experiments, ~ve assessed the frequency-response curve of the [AS smooth muscle strips to varying frequencies of EFS. The experiments were repeated in the presence of I x 10 -7 and 3 x 10 -7
715
0 Z 0 Z W I-U3
-20
'~/
0.5
1
2
5
I
I
I
I
O--O II--Ie 0--0
CONTROL G/4.ANIN GALANIN ( 3 X I O - / M )
(1XIO-!M)
<: - 4 0 Z ._1 ._1 I-Z W nW n
-6o
-80 -100
Figure 5. Influence of galanin on the decrease in the IAS tension caused by EFS. Galanin (3 × 1 0 - ' moUL) caused significant augmentation of the decrease in the IAS tension in response to different frequencies ofEFS (P < 0.05, n = 4).
716
CHAKDER AND RATTAN
0
ELECTRICAL FIELD STIMULATION 0,.5 1 2 5 --///
I
z O z h.I I-U')
I
[HZ]
I
rl--r-I CONTROL I - - I ¢AL~,N-(S-10) OXlO-6U)
-20
---< - 4 0
GASTROENTEROLOGY Vol. 100, No. 3
V~V
I
V--V
CONII~OL GALANIN-(15-29) (1X10-6M)
0--0
com~oL
~--4
GAI..,M41N-(7-16) (Ix10-SM)
z .._1 ._1 I.-Z 13.1
n,h.I {3_
-60
-80
Discussion The predominant effect of galanin on the resting tension of opossum I.AS is inhibitory. The inhibitory action of galanin on the IAS smooth muscle was surprising because in a large number of smooth muscle preparations including LES, galanin was primarily found to be contractile (1,2,11-14). The inhibitory effects of galanin on the IAS are exerted both at the myogenic as well as at the neural levels of the IAS smooth muscle. The myogenic action of galanin is suggested by a decrease in the IAS tension in response to galanin in the presence of the neurotoxin TTX. The concentration of TTX used was shown to abolish the neurally mediated/.AS relaxation (Figure 3).
-100 Figure 6. Influence of galanin-(1-10), galanin-(15-29), and galanin-(7-16) on EFS-induced decrease in the resting tension of the [AS. Galanin-(1-10) caused significant augmentation of the EFS responses (P < 0.05, n = 4). Galanin-(15-29) and galanin-(7-16), on the other hand, had no significant effect on the EFS responses o f the [AS (P > 0.05).
/.AS. Percent decrease in the IAS tension with 1 x 1 0 -7, 3 x 1 0 -7, and 1 x 10 -6 mol/L VIP in these experiments was 52 ± 5, 75 ± 6, and 90 ± 5, respectively, in control experiments. In the presence of galanin, these values were 42 ± 8%, 72 + 7%, and 87 ± 2°)o (P > 0.05;n = 4).
Effects of Galanin on the Resting Tension and on Neurally Mediated Relaxation of the Lower Esophageal Sphincter Galanin caused a concentration-dependent increase in the resting tension of the LES (Figure 7). Galanin (3 x 10 -6 mol/L) caused 47% ± 2% increase in the resting tension of the LES. Increase in the LES t~nsion caused by galanin was not significantly affected by the neurotoxin, TTX. The percent increase in the LES tension by galanin (1 x 10 -7 mol/L) before and after TTX were 28 ± 1 and 27 ± 3 respectively (P > 0.05; n = 4). The resting tensions in these experiments before and after TTX treatment were 1.88 ± 0.25 and 1.96 ± 0.27 g, respectively. On the other hand, galanin (1 x 10 -6 mol/L) caused a significant suppression of the percent decrease in the LES tension in response to EFS (Figure 7; P < 0.05). The percent decrease in the resting LES tension with 0.5 and 1 Hz in these experiments was 33 ± 2 and 65 ± 4; in the presence of galanin, these values were 15 ± 3 and 41 ± 6, respectively. This concentration of galanin had no significant effect on the resting tension of the LES; the values before and after galanin administration .were 2.07 ± 0.30 and 2.25 ± 0.30 g, respectively (P > 0.05, n = 5).
LOG [AGONIST] M -g 80
-8
7/,
'
I
-7 '
,
-6 '
-5
,
'
,
-4'
'
Z 0
Z W
o - - o GALANIN • • GALANIN-(1-10) ---* GALANIN-(15-29)
60
(/3 W
foT
.
z
40
,5
J
,., Z
m n.-°
20
~jo 7~. t
o..Ld
O/0
z
cco.
O z
-20
m "_J'
-40
~
-60
o n,-
-80
i
o,) °
-GALANIN(3XIO-='M) O - - o EFS (AFTERWASH)
L,,J
ILl n.
i
I 00
/!#
i
0.5
i
t
I
1
2
5
ELECTRICAL FIELD STIMULATION [HZ]
Figure 7. The influence of different concentrations of galanin on the resting tension of the LES {top) and on the decrease in the LES tension in response to different frequencies (Hz) of electrical field stimulation (bottom). Note that on the LES galanin-produced effects which were opposite to those in the I.AS. In the LES, galanin caused an increase in the resting tension; the concentration {3 x 10-' mol/L), which caused no significant increase in the LES tension, caused a significant suppression of the LES relaxation in response to EFS (P < 0.051. Note that the effect o f g a l a n i n in causing suppression of LES relaxation Was completely reversible to the control levels after the wash. Galanin-(1-10) and galanin-(15-29) produced no significmat effects on the resting tension of the LES. The effects of galanin fragments on EFSinduced decrease in the resting tension of the LES were not tested.
M a r c h 1991
The neural actions of galanin on opossum /AS consisted of (a) partial TTX-sensitive inhibitory effect of galanin in causing relaxation of the IAS smooth muscle strips and (b) augmentation of neurally mediated IAS relaxation. The augmentatory action of galanin was observed by the facilitation of the /AS relaxation caused by neural stimulation by EFS. The augmentatory action of galanin on neurally mediated response has been shown previously by Ohhashi and Jacobowitz (26]. The inhibitory effect of galanin on the I_AS is different from its excitatory effects on the LES and other gut tissues (1,5,14-15) but is similar to the inhibitory effect in different gastrointestinal preparations (13-17). Interestingly, also in the nonsphincteric smooth muscle strips obtained from the opossum rectum, galanin exerted inhibitory effects on the spontaneous phasic contractions. A significant decrease in the amplitude as well as the rate of contracion was observed (unpublished observations). The possibility was considered that the qualitative differences in the actions of galanin on two types of sphincteric tissues were relafed to in vivo and in vitro studies {19}. The present studies were performed on in vitro I.AS smooth muscle strips whereas the LES studies were carried out on the intact animals (11). Interestingly, when the LES studies were repeated on isolated smooth muscle strips, frank contraction (similar to that in vivo studies) was observed. These observations suggest that the actions of certain neuropeptides may not be generalized in all the gut organs. There are two major possibilities for the neurally mediated decrease in the resting tension of the I_AS in response to galanin and the augmentatory effect of galanin on EFS-induced IAS relaxation: (a) the release of an inhibitory neurotransmitter by galanin and (b) the augmentation of the inhibitory effect of the released inhibitory neurotransmitter. Vasoactive intestinal polypeptide has been suggested to be one of the inhibitory neurotransmitters of the IAS (20,21). The possible potentiation of the inhibitory effect of the released inhibitory neurotransmitter is unlikely, because the decrease in t h e / A S tension caused by VIP was not modified by galanin. Interestingly, studies in the isolated smooth muscle cells of the guinea pig small intestine suggest an augmentation of the inhibitory responses of VIP, isoproterenol, and dibutyryl 5'-cyclic adenosine monophosphate (cAMP} by galanin (27). Thus, it is possible that the augmentatory effects of galanin on the IAS is related to the release of inhibitory neurotransmitter substanae from the myenteric inhibitory neurons. Experiments designed to examine the possible release of a neurotransmitter from myenteric inhibitory neurons of the gut by galanin have not been performed. However, it has
M O T O R ACTIONS OF GALANIN
717
been suggested that in the hypothalamus, the galanininduced increase in the plasma levels of prolactin are mediated via the release of VIP (28}. The exact site of action of galanin in mediating a decreas6 in the resting tension of the IAS, the augmentation of the /AS relaxation by neural responses, and the cellular mechanisms responsible for the action of galanin on the IAS are not known presently. The direct excitatory and the !nhibitory effects of galanin and its fragments on the IAS smooth muscle are similar to their effects on other parts of the gut (5,11,15}. The direct and indirect potent actions of galanin and the demonstration of galanin-immunoreactive nerves in different layers of the entire gastrointestinal tract suggest a neurotransmitter or neuromodulatory role of galanin in the gut. However, studies providing direct evidence have not been performed. It appears that the presence of the N-terminal portion of galanin is important for the inhibitory effect of galanin on the I.AS, because the N-terminal fragment galanin-(1-10) produced only a decrease in the resting tension of the I.AS. Furthermore, galanin-(110) also caused an augmentation of the inhibitory responses to EFS on the I_AS. It is ir~teresting, however, that the decrease in the IAS tension by galanin(1-10) is considerably less than that produced by galanin. Specific studies investigating the actions of different lengths of N-terminal fragments would resolve this issue. It is of further interest that the C-terminal fragment, galanin-(15-29), did not cause any decrease in t h e / A S tension; instead, it caused a concentration-dependent increase in the IAS tension. Suggestions of the critical presence of the N-terminal portion of galanin (29,30) and it being responsible for the neural actions has been made before (14,31,32). The increase in the IAS tension may be caused by the direct effect of galanin-(15-29) on the /AS smooth muscle. The presence of galanin receptors on the/AS smooth muscle cells supports the concept of close proximity of galanin-containing nerve fibers on the gut smooth muscle (10). The actions of different fragments of galanin on the IAS smooth muscle strips are different from their effects on the guinea pig ileum and the iris sphincter (13). It was suggested that in these tissues the presence of the N terminal is primarily responsible for the direct smooth muscle contractile actions of galanin and that the neural actions of galanin reside in its C-terminal fragment; however, the effects of the C-terminal portion were not tested. Experiments dealing with the direct intracellular electrical recording from the myenteric neurons [33) and the actual release of the neurotransmitter (34) with different fragmentb of galanin may resolve the issue of structure-activity relationship and the critical importance of a particu-
718
CHAKDER AND RATTAN
lar fragment of galanin for a specific activity (neural vs. muscle). It is possible that the availability of selective antagonists of galanin provides further information on the nature of the galanin receptor or receptors and the physiological role of galanin in the gastrointestinal tract and the reason for the incomplete decrease in the IAS tension by galanin.
GASTROENTEROLOGY Vol. 100, No. 3
19.
20.
21.
22.
References 1. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V. Galanin--a novel biologically active peptide from porcine intestine. FEBS Lett 1963;164:124-128. 2. Rokaeus A, Melander T, Hokfelt T, Lundberg JM, Tatemoto K, Carlquist M, Mutt V. A galanin-like peptide in the central nervous system and intestine of the rat. Neurosci Lett 1964;47: 161-166. 3. Rokaeus A, Galanin: a newly isolated biologically active neuropeptide. Trends Neurosci 1967;10:156-164. 4. Sengupta A, Goyal R. Localization of galanin immunoreactivity in the opossum esophagus. J Auton Nerv Sys 1986;22:49-56. 5. Harling H, Messell T, Jansen SL, Hoist JJ, Poulsen SS. Occur' rence, distribution and motor effects of galanin in the porcine lower esophageal sphincter. Digestion 1969;42:151-157. 6. King SC, Slater P, Turnberg LA. Autoradiographic localization of binding sites for galanin and VIP in small intestine. Peptides 1989;10:313-317. 7. Ekblad E, Rokaeus A, Hakanson R, Sundler F. Galanin nerve fibers in the rat gut: distribution, origin and projections. Neuroscience 1985;16:355-363. 8. Gon~la T, Daniel EE, McDonald TJ, Fox JET, Brooks BD, Oki M. Distribution znd fui~ction of enteric GAL-IR nerves in dogs: comparison with VIP. Am J Physiol 1989;256:G864-G696. 9. Hoyle CHV, Burnstock G. Galanin-like immunoreactivity in enteric neurons of the human colon. J Anat 1989;166:23-33. 10~ Feher E, Burnstock G. Galanin-like immunoreactive nerve elements in the small intestine of the rat. An electron microscopic immunocytochemical study. Neurosci Lett 1988:92:137142. 11. Rattan S, Goyal RK. Effect of galanin on the opossum lower esophageal sphincter. Life Sci 1967;41:2783-2790. 12. Fontaine J, Lebrun P. Galanin: Ca2÷-dependent contractile effects on the isolated mouse distal colon. Eur J Pharmacol 1989;164:583-586. 13. Ekblad E, Hakanson R, Sundler F, Wahlestedt C. Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations. Br J Pharmacol 1985;86:241-246. 14. Fox JET, Brooks B, McDonald TJ, Barnett W, Kostolanska F, Yanaihara C, Yanaihara N, Rokaeus A. Actions of galanin fragments on rat, guinea-pig, and canine intestinal motility. Peptides 1966;9:1163-1189. 15. Fox JET, McDonald JT, Kostolanska F, Tatemoto K. Galanin: an inhibitory neural peptide of the canine small intestine. Life Sci 1966;39:103-110. 16. Allescher HD, Daniel EE, Dent J, Fox JET. Inhibitory function of VIP-PHI and galanin on canine pylorus. Am J Physio11989;256: G769-G797. 17. Bauer FE, Zintel A, Kenny MJ, Calder D, Ghatei MA, Bloom SR. Inhibitory effect of galanin on postprandial gastrointestinal motility and gut hormone release in humans. Gastroenterology 1969;97:260-264. 18. Muramatsu I, Yanaihara N. Contribution of galanin to non-
23.
24.
25. 26.
27.
26.
29.
30.
31.
32.
33.
34.
cholinergic, non-adrenergic transmission in rat ileum. Br J Pharmaco11988;94:1241-1249. Fox JET, Daniel EE, Jury J, Fox AE, Collins SM. Sites and mechanisms of action of neuropeptide in canine gastric motility differ in vivo and in vitro. Life Sci 1983;33:817-625. Nurko S, Rattan S. Role of vasoactive intestinal polypeptide in the internal anal sphincter relaxation of the opossum. J Clin Invest 1968;81:1146-1153. Biancani P, Walsh JH, Behar J. Vasoactive intestinal polypeptide: a neurotransmitter for relaxation of the rabbit internal anal sphincter. Gastroenterology 1965;89:667-874. Moummi C, Rattan S. Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation. Am J Physiol 1988;255:G571-G578. Rattan S, Moummi C. Influence of stimulators and inhibitors of cyclic nucleotides on lower esophageal sphincter. J Pharmacol Exp Ther 1989;248:703-709. Van Rossum JM. Cumulative dose-response curves. 11. Techniques for making dose-response curves in isolated organs and the evaluation of drug parameters. Arch lnt Pharmacodyn 1963;143:299-330. Zar JH. Biostatistical analysis. In: McElroy WD, Swanson CP, eds. Engelwood Cliffs, NJ: Prentice-Hall, 1974. Ohhashi T, Jacobowitz DM. Galanin potentiates electrical stimulation and exogenous norepinephrine-induced contractions in the rat vas deferens. Regul Pept 1985;12:163-171. Grider JR, Makhlouf GM. The modulatory action of galanin: potentiation of VIP-induced relaxation in isolated smooth muscle cells (abstr). Gastroenterology 1968:94:157. Inoue T, Kato Y, Koshiyama H, Yanaihara N, Imura H. Galanin stimulates the release of vasoactive intestinal peptide from perfused hypothalamic fragments in vitro and from periventricular structures into the cerebrospinal fluid in vivo in the rat. Neurosci Lett 1968;65:95-100. Amiranoff B, Lorinet A, Yanaihara N, Laburthe M. Structural requirement for galanin action in the pancreatic b cell line Rin m 5F. Eur J Pharmacol 1989:163:205-207. Hermansen K, Yanaihara N, Ahren B. On the nature of the galanin action on the endocrine pancreas: studies with six galanin fragments in the perfused dog pancreas. Acta Endocrinol 1969;121:545-550. Lagny-Pourmir I, Lorinet AM, Yanaihara N, Laburthe M. Structural requirement for galanin interaction with receptors from pancreatic Beta cells and from brain tissue of the rat. Peptides 1989;10:757-761. Fisone G, Berthold M, Bedecs K, Unden A, Bartfai T, Bartorelli R, Consolo S, Crawley J, Martin B. N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor. Proc Natl Acad Sci USA 1989;86:9568-9591. Tamura K, Palmer JM, Winkelmann CK, Wood JD. Mechanism of action of galanin on myenteric neurons. J Neurophysiol 1986;60:966-979. Yau WM, Dorsett JA, Youther ML. Evidence for galanin as an inhibitory neuropeptide on myenteric cholinergic neurons in the guinea pig small intestine. Neurosci Lett 1986:72:305-306.
Received March 15, 1990. Accepted August 28, 1990. Address requests for reprints to: Dr. Satish Rattan, Jefferson Medical College, Department of Medicine, Division of Gastroenterology and Hepatology, 901 College, 1025 Walnut Street, Philadelphia, Pennsylvania 19107. This work was supported by U.S. Publi c Health Service Grant DK-35385 from the National Institutes of Health. The authors thank Dr. B. S. Anand for valuable comments and Elenor Franco for typing the manuscript.
Effects of Galanin on the Opossum Internal Anal Sphincter: Structure-Activity Relationship S U S H A N T A CHAKDER a n d S A T I S H R A T T A N Division of Gastroenterologyand Hepatology,Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania
The present study was carried out to investigate the effects of porcine galanin-(1-29), N-terminal fragment galanin-(1-10), C-terminal fragment galanin(15-29), and the middle fragment galanin-(7-16) on the spontaneous tension of the opossum internal anal sphincter and on the decrease in the resting internal anal sphincter tension in response to neural stimulation by electrical field stimulation. Galanin and galanin-(1-10) caused a concentration-dependent decrease in the resting tension of internal anal sphincter and an augmentation of the percent decrease in the resting tension with electrical field stimulation. Galanin-(15-29), on the other hand, produced an increase in the resting tension of the internal anal sphincter and had no effect on the electrical field stimulation-induced decrease in the resting tension. Galanin-(7-16) produced no significant effect on the internal anal sphincter. The decrease in the internal anal sphincter tension b y galanin and galanin-(1-10) was partially antagonized by tetrodotoxin, whereas the increase in the internal anal sphincter tension caused by galanin(15-29) was not modified by tetrodotoxin. In contrast to its effect in the internal anal sphincter, galanin caused an increase in the resting tension and suppressed a decrease in the lower esophageal sphincter tension in response to electrical field stimulation. From these findings we conclude that (a) galanin exerts an inhibitory effect on.the internal anal sphincter by activating galanin receptors both at the intramural inhibitory neurons and at the internal anal sphincter smooth muscle and that the N-terminal portion of galanin m a y be responsible for these actions; (b) the contractile dction of galanin is produced by its action on the smooth muscle; and (c) the actions ofgalanin on the gastrointestinal tract are tissue s p e c i f i c .
alanin, a new neuropeptide with 29 amino acids, has been shown to be present in the central as G well as in the peripheral nervous system (1-3). In the gut, galanin has been shown to be widely distributed from the esophagus to the large intestine (4-9). It has been suggested that galanin is present in the myenteric and the Meissner's plexuses and" in the smooth muscle layers of the gut (6,7,9,10). Furthermore, galanin-immunoreactive fibers have been shown to be present in close proximity of the smooth muscle cells of the gut (10). Despite its wide distribution, the exact role of galanin in the gut is not clearly understood. It has been observed that galanin exerts both excitatory (1,5,11-14) and inhibitory effects on the motor functions of the gut (13-17). These actions may be the result of either a direct effect or may occur indirectly via the release of another neurotransmitter substance. Galanin has been suggested to be an excitatory neurotransmitter for certain components of the longitudinal smooth muscle contraction of the rat ileum (18). In the canine small intestine (15) and the pyloric sphincter (16), galanin has been suggested to act as an inhibitory neurotransmitter. Thus, galanin has been shown to have different effects on different parts of the gut. In some cases, the in vivo effects are different from those observed in vitro studies (19). However, the in vitro effects of galanin on the spontaneously tonic smooth muscles have not been examined. Recently, galanin immunoreactivity has also been demonstrated in the region of the canine internal anal sphincter where it coexists with vasoactive intestinal
Abbreviations used in this paper: IAS, internal anal sphincter; EFS, electrical field stimulation; TFX, tetrodotoxin. © 1991 by the American Gastroenterological Association 0016-5085/91/$3.00
712 CHAKDERAND RATTAN
p o l y p e p t i d e (VIP) [8). Previous studies on the internal anal s p h i n c t e r (/AS) suggest that VIP is one of the inhibitory n e u r o ~ a n s m i t t e r s ; also, the possibility of i n v o l v e m e n t of m u l t i p l e inhibitory n e u r o t r a n s m i t t e r s in the IAS has b e e n raised (20,21). T h e role of galanin in t h e / A S , however, is not k n o w n . Consequently, the p u r p o s e of the p r e s e n t investigation w a s to s t u d y the influence of galanin and its fragments o n the resting tone of the /AS a n d on neurally m e d i a t e d IAS relaxation. M a t e r i a l s a n d Methods
Preparation of Smooth Muscle Strips Opossums of either sex were killed by exsanguination after pentobarbital anesthesia (40 mg/kg IP). The entire anal canal along with a part of the rectum was isolated and transferred to a dissecting tray containing oxygenated I(rebs physiological solution. The composition of the Krebs solution (in mmolJL) was as follows: NaCI, 118.07; KCI, 4.69; CaC12, 2.52; MgSO 4, 1.16; NaH2PO 4, 1.01; NaHCO3, 25; and glucose, 11.10. • The anal canal was freed from extraneous tissues of blood vessels, nerves, connective tissue, fascia, and the external anal sphincter skeletal muscle by sharp dissection. The anal canal was then opened by an incision along the longitudinal axis and pinned flat on the dissecting tray with the mucosal side up. The mucosa was removed by gentle scraping with fine scissors and forceps, starting from the uppermost part of the anal canal. Circular smooth muscle strips (approximately 2 mm wide and 1 cm long) were obtained from the circumference of the anal canal. The ends of the muscle strips were tied with 4-0 silk suture.
Recording of Isometric Tension The smooth muscle strips were transferred to jacketed 2-mL tissue baths filled with Krebs solution, which was maintained at 34°C and equilibrated with 95% O2 5% CO2, and were anchored to the muscle bath at the lower end and isometric muscle transducers (model FTO3; Grass Instruments Co., Quincy, MA) at the other end. The isometric tension of the smooth muscle strips at rest as Well as that in response to different stimuli were recorded on a Beckman Dynograph Recorder (model R411; Beckman Instruments, Schiller Park, IL). After an equilibration period of 1 hour, the optimal length and the baseline of each /AS smooth muscle strip was determined according to a previously described method (22). The smooth muscle strips of the lower esophageal sphincter (LES) were prepared in the same manner according to a previously described method (23).
Electrical Field Stimulation Once the plateau of spontaneous/AS or LES tension was achieved, the resting tension of each smooth muscle strip was determined by supramaximal electrical field stim-
GASTROENTEROLOGYVol. 100, No. 3
ulation (EFS) (30 V, 20 Hz, 0.5 ms pulse duration, for 4 seconds). The response to EFS (20-30 V, 0.5 n~ pulse duration, for 4 seconds) at varying frequencies (0.5-5 Hz) on the resting tension of the/AS strips in the presence and absence of various agents was examined. The electrodes used consisted of thin platinum wires lying parallel to the smooth muscle strips; the EFS was delivered by a Grass stimulator (model $48; Grass Instruments).
Drug Responses The effect of different agents on the resting tension of the IAS and on the decrease in the/AS tension by EFS was investigated. The response to different concentrations of the agonists on the resting IAS tension was examined using cumulative concentration responses (24). After the concentration-response curves had been derived, the smooth muscle strips were washed six times, the resting tension was allowed to recover to the pretreatment level, and an additional 30-minute rest was given before the next agonist concentration-response curve was derived. The responses to different agents and stimuli that caused a decrease in the resting tension of the IAS were quantified as percentage of maximal decrease in the/AS tension by supramaximal EFS. The responses of agents which caused an increase in the [AS tension were quantified as the percentage of maximal rise in the tension by the maximal concentration ofbethanechol (1 x 10 -~ mol/L). In the case of antagonists or the neurotoxin, tetrodotoxin, a precalculated amount of the agent was added in the bath to achieve the desired final concentration. The concentrations of the different antagonists used have been previously tested to be appropriate against their respective agonists and stimuli. The concentration-response curves of the respective agonists and the frequancy-response curves were examined before and after the addition of galanin and its fragments.
Drugs The following drugs were used: galanin (porcine, mol wt 3211.03) (Peninsula Laboratories, Inc., Belmont, CA]; galanin 1-10 (mol wt 1081.31): galanin-(7-161 (mol wt 1011.31); galanin-(15-29) (mol wt 1711.09) (synthesized in the Protein Chemistry Laboratory, University of Florida, Gainesville, FL); tetrodotoxin (TTX, mol wt 319.28) (Calbiochem, San Diego, CA); propranolol hydrochloride (tool wt 292.34) (Ayerst Laboratories, New York, NY); atropine sulphate (mol wt 694.82); hexamethoninm chloride (mol wt 273.28); and VIP (porcine, mol wt 3326) (Sigma Chemical Co., St. Louis, MO). All agents were dissolved and diluted in Krebs solution. The solutions of different agents used were freshly prepared on the day of the experiment, and the volume of solutions added to the muscle baths was not allowed to exceed 0.1 mL. The vials, glassware, pipette tips, and the muscle baths were pretreated with 2.5% bovine serum albumin.
March 1991
MOTOR ACTIONS OF GALANIN 713
Data Analysis All changes in the resting (AS tension in response to
100
drugs and EFS were expressed on the bases of percent and absolute changes in a given smooth muscle strip before find after different pretreatments. All the values were expressed as mean -+ SE of different experiments. For comparison purposes, the responses to the agonists and EFS, before and after the use of antagonists, were obtained from the same smooth muscle strip. Thus each [AS smooth muscle strip served as its own control. The statistical significance of group differences was analyzed using Student's t test (25).
Z W F--
+/
W
Z W (.~ n~ w o_
Effects of Galanin and its Fragments on the Resting Tension of Internal Anal Sphincter
Tetrodotoxin (1 x 10 -6 tool/L), which abolished the decrease in the IAS tension in response to
]
60
Results
Influence of Tetrodotoxin on the Inhibitory Effects of Galanin on the Resting Tension of Internal Anal Sphb~cter
80
Z
n~
The amino acid sequences of galanin and the different fragments used in the present study are given in Table 1. Galanin and the N-terminal fragment, galanin-(110), caused a concentration-dependent decrease in the resting tension of the IAS. On the other hand, galanin-(15-29), the C-termfnal fragment of galanin, caused an increase in the IAS tension (Figures 1 and 2). The maximal decrease in the I_AStension observed with galanin (3 x 10 -~ mol/L) was 51% + 3%. The maximal decrease with the highest concentration of galanin-(1-10) tested (1 x 10 -4 mol/L) was only 24% _+ 2%. Higher concentrations of galanin-(1-10) were not tested because of solubility problems. The middle fragment of the galanin molecule, galanin-(7-16), produced variable effects on the resting tension of the I.AS. Of the seven smooth muscle strips examined, four produced no response, two showed contraction, and in one the response was biphasic (relaxation followed by contraction).
A - - A GALANIN-(I5-29)
Z 0
40
+/
O ~ O ~ Z 0 oo Z w
O
-20 ~T
~O 0
o~
-40
~o~ 6
Z ._J _J F-Z W C.) nl W 8_
A
A
,/
20
If', A
O--O CALANIN
-60
e--o
0ALANIN-0-10)
-80 -100
//
I
-7
l
I
,
-6
I
-5
~
I
-4
LOG [AGONIST] M Figure 1. Concentration-effect curves showing the effects ofgalanin and its different fragments. Each point represents mean -- SE of four observations obtained from different animals. The absence of the SE bars in certain points in this and the subsequent figures is caused by their small size and loss in the symbols in computerized graphs. Galanin and galanin-(1-10l caused a concentrationdependent decrease in the resting tension of the IAS. Galanin(15-29), on the other hand, caused a concentration-dependent increase in the resting tension of the [AS. Galanin-(7-16) had no reproducible effect on the resting IAS tension (not shown in the figure).
Table 1. Amino Acid Sequences of Galanin and Its Fragments Galanin
G•y-Trp•Thr-Leu-Asn•Ser-A•a•G•y-Tyr•Leu-Leu•G•y•Pr••His•A•a-lIe-Asp-Asn•His•Arg-Ser-Phe•His-Asp-Lys-Tyr•G•y-Leu-A•a•NH Galanin-(1-10)
Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu Galanin-(7-16)
Ala-Gly-Tyr-L[u-Leu-Gly-Pro-His-Ala-Ile Galanin-(15-29)
Ala-Ile-Asp-Asn-His-Axg-Ser-Phe-His-Asp-Lys-Tyr-Gly-Leu-Ala-NH2
2
714
CHAKDER AND RATTAN
GASTROENTEROLOGY Vol. 100, No. 3
Galanin
2_
(M)
2
o ~
o
~
•
•
•
•
o
,
&
Galanin-(I-10)
3 ,nin f
control
~0
• GO).
'
"~,
(M) I
,~
I
.5
I
EFS
I
I
2
5
(I-:IZ)
Galanin ~
Ot
•
Galanin-(I 5-29)
Figure 2. Typical responses of galanin a n d its different fragments on the resting tension of the [AS (left) and on the decrease in the [AS tens i o n in r e s p o n s e to EFS (right). Galanin and galanin(1-10) caused a concentration-dependent decrease, whereas galanin-(15-29) caused an increase in the [AS tension.
(M)
.~..4
0 .5 1
OL-.
EFS
:i:ii" _
,
2
5
(HZ)
--
neural stimulation, caused partial but significant ddcrease in the inhibitory responses to galanin (Figure 3). The inhibitory response of galanin on the IAS were not modified by VIP or calcitonin gene-related peptide (CGRP) antagonists (unpublished observations). As with galanin, the decrease in the resting tension of the IAS produced by galanin-(1-10) was also partially but significantly suppressed by the neurotoxin. The decrease in the resting tension of the IAS with I x 10 -s and 3 x 10 -s mol/L of galanin-(1-10)in the control experiments was 12% _+ 2% and 19% _ 2%, respectively. In the presence of TTX these values were 5% _ 1% and 9% _ 2%, respectively (P < 0.05 for both; n = 4).
Influence of Atropine, Hexamethonium, and Propranolol on the Inhibitory Effects of Galanin on the Resting Tension of the Internal Anal Sphincter The muscarinic antagonist atropine (1 x 10 -s mol/L) had no significant influence on the inhibitory effects of galanin and galanin-(1-10) on the resting tension of the IAS smooth mt~scle strips (Figure 4).
ELECTRICAL FIELD STIMULATION [HZ] 0.5 0 ~/J--
"~
z
1 .L
~,~
2 ~
5 .1.
, --
10 3.
== EFS (l'l'X)
~(5 "e
o
\o~\i
\6
-20
,,,z 1-03 ~-- - 4 0 z
-_.I ,_I
fi
OALANIN (TI'X)
X ~
0"--(5
OALANIN (CONTROL)
T n
-6o
z w
[]
(3wn.-80 0..
n
-100
/i/
I -7
,
EFS (CONTROL)
I , ~x13 , -6 -5 LOG [GALANIN] M
I -4
Figure 3. Influence of Trx on the inhibitory responses ofgalanin on the IAS. Tetrodotoxin in a concentration that abolished EFS-mediated decrease in the [AS tension resulted in a significant antagonism of the decrease in the IAS tension caused by galanin ( P < o.os; n = 4).
March 1991
MOTOR ACTIONS OF GALANIN
GALANIN
GALANIN-(I-IO)
Ox~O-eM) (3XlO-eM) OXlO-~M) (3Xlo-SM)
1
Z
o Z W I--
_<
-20
Z _1 ._1
~_ - 4 0 Z W
CONTROL ATROPINE (1X10-5M)
(3 W
-60 Figure 4. Influence of atropine on the decrease in the IAS tension caused by galanin and galanin-(1-10). Atropine failed to cause a
significant modification of the responses of galanin and gnlanin(1-10) (P > 0.05, n = 4).
The nicotinic antagonist hexamethonium (1 x 10 -6 mol/L), and ~-adrenoceptor antagonist propranolol (1 x 10 -6 mol/L) also had no significant effect on the decrease in the [AS tension caused by galanin. In this series of experiments, the decrease in the resting tension of the [AS with 1 x 10 -6 and 3 x 10 -6 mol/L galanin was 14% _+ 2% and 31% -+ 4%, respectively, in the controls; after the administration of hexamethonium and propranolol, the corresponding values were 13% -+ 2% and 30% -+ 3% and 11% - 2% and 29% +6%, respectively. These values were not significantly different from the control values (P > 0.05; n = 4).
mol/L galanin. Interestingly, the percent decrease in the resting tension in response to EFS in the presence of galanin was significantly greater than the decrease during control EFS. The quantitative data is shown in Figure 5. Galanin (3 x 10 -7 tool/L) caused a significant augmentation in the decrease in the [AS tension caused by 0.5 and 1 Hz of EFS from 42% _+ 2% and 59% _+ 5% to 68% +- 5% and 79% _ 4%, respectively, (P < 0.05; n = 4). The resting tension in these experiments before and after galanin was 1.85 _+ 0.28 and 1.50 _ 0.15 g, respectively. Galanin-(1-10) had an effect similar to galanin in causing an augmentation of EFS-induced decrease in the resting tension of the IAS smooth muscle (Figure 6). However, galanin-(15-29) and galanin-(7-16) had no significant effect on the decrease in the resting [AS tension in response to EFS (Figure 6). An actual tracing showing augmentation of the [AS responses to EFS by galanin has been included in Figure 2.
Influence of Galanin on the Decrease in the Resting Tension of the Internal Anal Sphincter Smooth Muscle by Vasoactive Intestinal Polypeptide To test the possibility that the augmentation of neurally mediated [AS relaxation is via the potentiation of the inhibitory effect of the released inhibitory neurotransmitter, we tested the influence of galanin on the percent decrease in the [AS tension by one of the candidates of inhibitory neurotransmitters, VIP. Galanin did not cause any significant change in VIP-induced decrease in the resting tension of the ELECTRICAL FIELD STIMULATION [HZ]
Influence of Tetrodotoxin and Atropine on the Increase in the Internal Anal Sphincter Tension Caused by Galanin-(15-29) The neurotoxin TTX and the muscarinic antagonist atropine had no significant effect on the resting tension of the [AS smooth muscle. The increase in the [AS tension with galanin-(15-29), 3 x 10 -6 mol/L and 3 x 10 -s mol/L, was 27% +- 6% and 53% _ 7% before and 23% - 8% and 41% _ 8% after TTX, respectively, (P > 0 . 0 5 ; n = 5).
Influence of Galanin and Galanin Fragments on the Relaxation of the Internal Anal Sphincter Smooth Muscles by Neural Stimulation In this set of experiments, ~ve assessed the frequency-response curve of the [AS smooth muscle strips to varying frequencies of EFS. The experiments were repeated in the presence of I x 10 -7 and 3 x 10 -7
715
0 Z 0 Z W I-U3
-20
'~/
0.5
1
2
5
I
I
I
I
O--O II--Ie 0--0
CONTROL G/4.ANIN GALANIN ( 3 X I O - / M )
(1XIO-!M)
<: - 4 0 Z ._1 ._1 I-Z W nW n
-6o
-80 -100
Figure 5. Influence of galanin on the decrease in the IAS tension caused by EFS. Galanin (3 × 1 0 - ' moUL) caused significant augmentation of the decrease in the IAS tension in response to different frequencies ofEFS (P < 0.05, n = 4).
716
CHAKDER AND RATTAN
0
ELECTRICAL FIELD STIMULATION 0,.5 1 2 5 --///
I
z O z h.I I-U')
I
[HZ]
I
rl--r-I CONTROL I - - I ¢AL~,N-(S-10) OXlO-6U)
-20
---< - 4 0
GASTROENTEROLOGY Vol. 100, No. 3
V~V
I
V--V
CONII~OL GALANIN-(15-29) (1X10-6M)
0--0
com~oL
~--4
GAI..,M41N-(7-16) (Ix10-SM)
z .._1 ._1 I.-Z 13.1
n,h.I {3_
-60
-80
Discussion The predominant effect of galanin on the resting tension of opossum I.AS is inhibitory. The inhibitory action of galanin on the IAS smooth muscle was surprising because in a large number of smooth muscle preparations including LES, galanin was primarily found to be contractile (1,2,11-14). The inhibitory effects of galanin on the IAS are exerted both at the myogenic as well as at the neural levels of the IAS smooth muscle. The myogenic action of galanin is suggested by a decrease in the IAS tension in response to galanin in the presence of the neurotoxin TTX. The concentration of TTX used was shown to abolish the neurally mediated/.AS relaxation (Figure 3).
-100 Figure 6. Influence of galanin-(1-10), galanin-(15-29), and galanin-(7-16) on EFS-induced decrease in the resting tension of the [AS. Galanin-(1-10) caused significant augmentation of the EFS responses (P < 0.05, n = 4). Galanin-(15-29) and galanin-(7-16), on the other hand, had no significant effect on the EFS responses o f the [AS (P > 0.05).
/.AS. Percent decrease in the IAS tension with 1 x 1 0 -7, 3 x 1 0 -7, and 1 x 10 -6 mol/L VIP in these experiments was 52 ± 5, 75 ± 6, and 90 ± 5, respectively, in control experiments. In the presence of galanin, these values were 42 ± 8%, 72 + 7%, and 87 ± 2°)o (P > 0.05;n = 4).
Effects of Galanin on the Resting Tension and on Neurally Mediated Relaxation of the Lower Esophageal Sphincter Galanin caused a concentration-dependent increase in the resting tension of the LES (Figure 7). Galanin (3 x 10 -6 mol/L) caused 47% ± 2% increase in the resting tension of the LES. Increase in the LES t~nsion caused by galanin was not significantly affected by the neurotoxin, TTX. The percent increase in the LES tension by galanin (1 x 10 -7 mol/L) before and after TTX were 28 ± 1 and 27 ± 3 respectively (P > 0.05; n = 4). The resting tensions in these experiments before and after TTX treatment were 1.88 ± 0.25 and 1.96 ± 0.27 g, respectively. On the other hand, galanin (1 x 10 -6 mol/L) caused a significant suppression of the percent decrease in the LES tension in response to EFS (Figure 7; P < 0.05). The percent decrease in the resting LES tension with 0.5 and 1 Hz in these experiments was 33 ± 2 and 65 ± 4; in the presence of galanin, these values were 15 ± 3 and 41 ± 6, respectively. This concentration of galanin had no significant effect on the resting tension of the LES; the values before and after galanin administration .were 2.07 ± 0.30 and 2.25 ± 0.30 g, respectively (P > 0.05, n = 5).
LOG [AGONIST] M -g 80
-8
7/,
'
I
-7 '
,
-6 '
-5
,
'
,
-4'
'
Z 0
Z W
o - - o GALANIN • • GALANIN-(1-10) ---* GALANIN-(15-29)
60
(/3 W
foT
.
z
40
,5
J
,., Z
m n.-°
20
~jo 7~. t
o..Ld
O/0
z
cco.
O z
-20
m "_J'
-40
~
-60
o n,-
-80
i
o,) °
-GALANIN(3XIO-='M) O - - o EFS (AFTERWASH)
L,,J
ILl n.
i
I 00
/!#
i
0.5
i
t
I
1
2
5
ELECTRICAL FIELD STIMULATION [HZ]
Figure 7. The influence of different concentrations of galanin on the resting tension of the LES {top) and on the decrease in the LES tension in response to different frequencies (Hz) of electrical field stimulation (bottom). Note that on the LES galanin-produced effects which were opposite to those in the I.AS. In the LES, galanin caused an increase in the resting tension; the concentration {3 x 10-' mol/L), which caused no significant increase in the LES tension, caused a significant suppression of the LES relaxation in response to EFS (P < 0.051. Note that the effect o f g a l a n i n in causing suppression of LES relaxation Was completely reversible to the control levels after the wash. Galanin-(1-10) and galanin-(15-29) produced no significmat effects on the resting tension of the LES. The effects of galanin fragments on EFSinduced decrease in the resting tension of the LES were not tested.
M a r c h 1991
The neural actions of galanin on opossum /AS consisted of (a) partial TTX-sensitive inhibitory effect of galanin in causing relaxation of the IAS smooth muscle strips and (b) augmentation of neurally mediated IAS relaxation. The augmentatory action of galanin was observed by the facilitation of the /AS relaxation caused by neural stimulation by EFS. The augmentatory action of galanin on neurally mediated response has been shown previously by Ohhashi and Jacobowitz (26]. The inhibitory effect of galanin on the I_AS is different from its excitatory effects on the LES and other gut tissues (1,5,14-15) but is similar to the inhibitory effect in different gastrointestinal preparations (13-17). Interestingly, also in the nonsphincteric smooth muscle strips obtained from the opossum rectum, galanin exerted inhibitory effects on the spontaneous phasic contractions. A significant decrease in the amplitude as well as the rate of contracion was observed (unpublished observations). The possibility was considered that the qualitative differences in the actions of galanin on two types of sphincteric tissues were relafed to in vivo and in vitro studies {19}. The present studies were performed on in vitro I.AS smooth muscle strips whereas the LES studies were carried out on the intact animals (11). Interestingly, when the LES studies were repeated on isolated smooth muscle strips, frank contraction (similar to that in vivo studies) was observed. These observations suggest that the actions of certain neuropeptides may not be generalized in all the gut organs. There are two major possibilities for the neurally mediated decrease in the resting tension of the I_AS in response to galanin and the augmentatory effect of galanin on EFS-induced IAS relaxation: (a) the release of an inhibitory neurotransmitter by galanin and (b) the augmentation of the inhibitory effect of the released inhibitory neurotransmitter. Vasoactive intestinal polypeptide has been suggested to be one of the inhibitory neurotransmitters of the IAS (20,21). The possible potentiation of the inhibitory effect of the released inhibitory neurotransmitter is unlikely, because the decrease in t h e / A S tension caused by VIP was not modified by galanin. Interestingly, studies in the isolated smooth muscle cells of the guinea pig small intestine suggest an augmentation of the inhibitory responses of VIP, isoproterenol, and dibutyryl 5'-cyclic adenosine monophosphate (cAMP} by galanin (27). Thus, it is possible that the augmentatory effects of galanin on the IAS is related to the release of inhibitory neurotransmitter substanae from the myenteric inhibitory neurons. Experiments designed to examine the possible release of a neurotransmitter from myenteric inhibitory neurons of the gut by galanin have not been performed. However, it has
M O T O R ACTIONS OF GALANIN
717
been suggested that in the hypothalamus, the galanininduced increase in the plasma levels of prolactin are mediated via the release of VIP (28}. The exact site of action of galanin in mediating a decreas6 in the resting tension of the IAS, the augmentation of the /AS relaxation by neural responses, and the cellular mechanisms responsible for the action of galanin on the IAS are not known presently. The direct excitatory and the !nhibitory effects of galanin and its fragments on the IAS smooth muscle are similar to their effects on other parts of the gut (5,11,15}. The direct and indirect potent actions of galanin and the demonstration of galanin-immunoreactive nerves in different layers of the entire gastrointestinal tract suggest a neurotransmitter or neuromodulatory role of galanin in the gut. However, studies providing direct evidence have not been performed. It appears that the presence of the N-terminal portion of galanin is important for the inhibitory effect of galanin on the I.AS, because the N-terminal fragment galanin-(1-10) produced only a decrease in the resting tension of the I.AS. Furthermore, galanin-(110) also caused an augmentation of the inhibitory responses to EFS on the I_AS. It is ir~teresting, however, that the decrease in the IAS tension by galanin(1-10) is considerably less than that produced by galanin. Specific studies investigating the actions of different lengths of N-terminal fragments would resolve this issue. It is of further interest that the C-terminal fragment, galanin-(15-29), did not cause any decrease in t h e / A S tension; instead, it caused a concentration-dependent increase in the IAS tension. Suggestions of the critical presence of the N-terminal portion of galanin (29,30) and it being responsible for the neural actions has been made before (14,31,32). The increase in the IAS tension may be caused by the direct effect of galanin-(15-29) on the /AS smooth muscle. The presence of galanin receptors on the/AS smooth muscle cells supports the concept of close proximity of galanin-containing nerve fibers on the gut smooth muscle (10). The actions of different fragments of galanin on the IAS smooth muscle strips are different from their effects on the guinea pig ileum and the iris sphincter (13). It was suggested that in these tissues the presence of the N terminal is primarily responsible for the direct smooth muscle contractile actions of galanin and that the neural actions of galanin reside in its C-terminal fragment; however, the effects of the C-terminal portion were not tested. Experiments dealing with the direct intracellular electrical recording from the myenteric neurons [33) and the actual release of the neurotransmitter (34) with different fragmentb of galanin may resolve the issue of structure-activity relationship and the critical importance of a particu-
718
CHAKDER AND RATTAN
lar fragment of galanin for a specific activity (neural vs. muscle). It is possible that the availability of selective antagonists of galanin provides further information on the nature of the galanin receptor or receptors and the physiological role of galanin in the gastrointestinal tract and the reason for the incomplete decrease in the IAS tension by galanin.
GASTROENTEROLOGY Vol. 100, No. 3
19.
20.
21.
22.
References 1. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V. Galanin--a novel biologically active peptide from porcine intestine. FEBS Lett 1963;164:124-128. 2. Rokaeus A, Melander T, Hokfelt T, Lundberg JM, Tatemoto K, Carlquist M, Mutt V. A galanin-like peptide in the central nervous system and intestine of the rat. Neurosci Lett 1964;47: 161-166. 3. Rokaeus A, Galanin: a newly isolated biologically active neuropeptide. Trends Neurosci 1967;10:156-164. 4. Sengupta A, Goyal R. Localization of galanin immunoreactivity in the opossum esophagus. J Auton Nerv Sys 1986;22:49-56. 5. Harling H, Messell T, Jansen SL, Hoist JJ, Poulsen SS. Occur' rence, distribution and motor effects of galanin in the porcine lower esophageal sphincter. Digestion 1969;42:151-157. 6. King SC, Slater P, Turnberg LA. Autoradiographic localization of binding sites for galanin and VIP in small intestine. Peptides 1989;10:313-317. 7. Ekblad E, Rokaeus A, Hakanson R, Sundler F. Galanin nerve fibers in the rat gut: distribution, origin and projections. Neuroscience 1985;16:355-363. 8. Gon~la T, Daniel EE, McDonald TJ, Fox JET, Brooks BD, Oki M. Distribution znd fui~ction of enteric GAL-IR nerves in dogs: comparison with VIP. Am J Physiol 1989;256:G864-G696. 9. Hoyle CHV, Burnstock G. Galanin-like immunoreactivity in enteric neurons of the human colon. J Anat 1989;166:23-33. 10~ Feher E, Burnstock G. Galanin-like immunoreactive nerve elements in the small intestine of the rat. An electron microscopic immunocytochemical study. Neurosci Lett 1988:92:137142. 11. Rattan S, Goyal RK. Effect of galanin on the opossum lower esophageal sphincter. Life Sci 1967;41:2783-2790. 12. Fontaine J, Lebrun P. Galanin: Ca2÷-dependent contractile effects on the isolated mouse distal colon. Eur J Pharmacol 1989;164:583-586. 13. Ekblad E, Hakanson R, Sundler F, Wahlestedt C. Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations. Br J Pharmacol 1985;86:241-246. 14. Fox JET, Brooks B, McDonald TJ, Barnett W, Kostolanska F, Yanaihara C, Yanaihara N, Rokaeus A. Actions of galanin fragments on rat, guinea-pig, and canine intestinal motility. Peptides 1966;9:1163-1189. 15. Fox JET, McDonald JT, Kostolanska F, Tatemoto K. Galanin: an inhibitory neural peptide of the canine small intestine. Life Sci 1966;39:103-110. 16. Allescher HD, Daniel EE, Dent J, Fox JET. Inhibitory function of VIP-PHI and galanin on canine pylorus. Am J Physio11989;256: G769-G797. 17. Bauer FE, Zintel A, Kenny MJ, Calder D, Ghatei MA, Bloom SR. Inhibitory effect of galanin on postprandial gastrointestinal motility and gut hormone release in humans. Gastroenterology 1969;97:260-264. 18. Muramatsu I, Yanaihara N. Contribution of galanin to non-
23.
24.
25. 26.
27.
26.
29.
30.
31.
32.
33.
34.
cholinergic, non-adrenergic transmission in rat ileum. Br J Pharmaco11988;94:1241-1249. Fox JET, Daniel EE, Jury J, Fox AE, Collins SM. Sites and mechanisms of action of neuropeptide in canine gastric motility differ in vivo and in vitro. Life Sci 1983;33:817-625. Nurko S, Rattan S. Role of vasoactive intestinal polypeptide in the internal anal sphincter relaxation of the opossum. J Clin Invest 1968;81:1146-1153. Biancani P, Walsh JH, Behar J. Vasoactive intestinal polypeptide: a neurotransmitter for relaxation of the rabbit internal anal sphincter. Gastroenterology 1965;89:667-874. Moummi C, Rattan S. Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation. Am J Physiol 1988;255:G571-G578. Rattan S, Moummi C. Influence of stimulators and inhibitors of cyclic nucleotides on lower esophageal sphincter. J Pharmacol Exp Ther 1989;248:703-709. Van Rossum JM. Cumulative dose-response curves. 11. Techniques for making dose-response curves in isolated organs and the evaluation of drug parameters. Arch lnt Pharmacodyn 1963;143:299-330. Zar JH. Biostatistical analysis. In: McElroy WD, Swanson CP, eds. Engelwood Cliffs, NJ: Prentice-Hall, 1974. Ohhashi T, Jacobowitz DM. Galanin potentiates electrical stimulation and exogenous norepinephrine-induced contractions in the rat vas deferens. Regul Pept 1985;12:163-171. Grider JR, Makhlouf GM. The modulatory action of galanin: potentiation of VIP-induced relaxation in isolated smooth muscle cells (abstr). Gastroenterology 1968:94:157. Inoue T, Kato Y, Koshiyama H, Yanaihara N, Imura H. Galanin stimulates the release of vasoactive intestinal peptide from perfused hypothalamic fragments in vitro and from periventricular structures into the cerebrospinal fluid in vivo in the rat. Neurosci Lett 1968;65:95-100. Amiranoff B, Lorinet A, Yanaihara N, Laburthe M. Structural requirement for galanin action in the pancreatic b cell line Rin m 5F. Eur J Pharmacol 1989:163:205-207. Hermansen K, Yanaihara N, Ahren B. On the nature of the galanin action on the endocrine pancreas: studies with six galanin fragments in the perfused dog pancreas. Acta Endocrinol 1969;121:545-550. Lagny-Pourmir I, Lorinet AM, Yanaihara N, Laburthe M. Structural requirement for galanin interaction with receptors from pancreatic Beta cells and from brain tissue of the rat. Peptides 1989;10:757-761. Fisone G, Berthold M, Bedecs K, Unden A, Bartfai T, Bartorelli R, Consolo S, Crawley J, Martin B. N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor. Proc Natl Acad Sci USA 1989;86:9568-9591. Tamura K, Palmer JM, Winkelmann CK, Wood JD. Mechanism of action of galanin on myenteric neurons. J Neurophysiol 1986;60:966-979. Yau WM, Dorsett JA, Youther ML. Evidence for galanin as an inhibitory neuropeptide on myenteric cholinergic neurons in the guinea pig small intestine. Neurosci Lett 1986:72:305-306.
Received March 15, 1990. Accepted August 28, 1990. Address requests for reprints to: Dr. Satish Rattan, Jefferson Medical College, Department of Medicine, Division of Gastroenterology and Hepatology, 901 College, 1025 Walnut Street, Philadelphia, Pennsylvania 19107. This work was supported by U.S. Publi c Health Service Grant DK-35385 from the National Institutes of Health. The authors thank Dr. B. S. Anand for valuable comments and Elenor Franco for typing the manuscript.